Techniques for covalent immobilization of molecules on surfaces are of crucial importance in surface science. For instance immobilized enzymes possess many benefits which include that no or only extremely small amount of immobilized enzyme dissolves in the reaction. Upon completion, reaction mixtures typically contain only solvent and reaction products. The immobilized enzyme is easily removed from the reaction making it easy to recycle.
Examples include but are not limited to areas such as catalysis, biosensors, microcontact printing, chromatography, and analytical devices. Several immobilization techniques are available today, for instance based on silanol chemistry, click chemistry, and one commonly used method for immobilization of biomolecules, the so called NHS (N-hydroxysuccinimid) method. Drawbacks for all those techniques include unstable bonds between immobilized molecules and the surface, expensive toxic chemicals or a prerequisite for organic solvents during the introduction of reactive structures on the surfaces.
The most used technique today is the so called NHS (N-hydroxysuccinimide) coupling technique. One of several disadvantages with this technique has been described by e.g. Wilchek. According to the latter author, this method is known to yield unstable bonds especially for single point attached molecules. It has e.g. been reported that 50% of the alanine immobilized by this method can be lost in 40 days (Cuatrecasas et al. in Biochemistry, vol. 11, p. 2291, 1972). According to Wilchek et al. in Biochemistry, vol. 26, p. 2155, 1987 the standard procedure to prepare NHS esters (namely N-hydroxysuccinimide and carbodiimides) leads to the formation of unstable immobilized compounds on polymers that also contain hydroxyl groups. This phenomenon is due to the formation of a p-alanine derivative which binds to the hydroxy-containing polymer, resulting in an unstable bond.
Other disadvantages of the NHS technique include that the ester bond which is used for covalent immobilization of molecules such as alanine or IgG will compete with the hydrolysis of the ester in aqueous media and that anhydrous conditions must be used in some steps for preparation of this final ester bond which involves use of dioxane. Step 1 in this process is the immobilization of a diamine (3,′3 diaminodipropylcarbodiimide) to the matrix followed in step 2 by intense washing with dioxane to be able to create anhydrous conditions for the next step 3. In step 3, N-hydroxysuccinimid is added together with the matrix and 3,′3-diaminodipropylamine which reacts with the introduced amine group on the surface in dioxane.
Alternatives to the NHS method exist but the reaction conditions involve use of organic solvents and expensive chemicals.
Pavlovic et al. used electro contact printing to immobilize proteins in patterns on a thiolated flat silicon surface, by site-selective oxidation of thiols to thiolsulphinates (Nanoletters vol. 3, No. 6, 779-781, 2003).
WO 2009/074692 discloses a method for partially derivatizing a curved surface comprising electro-oxidation.